This invention relates to the field of lipsomal immunoassays, and particularly to new-substrates for immunoassays utilizing .beta.-galactosidase.
In the assay of digoxin and other clinically important low concentration analytes which may be present in serum, especially human serum such as blood, using .beta.-galactosidase-encapsulated liposomes, it is important to utilize an extremely sensitive chromogenic substrate in order to achieve the essential assay dynamic range. This is especially true when such an assay is performed on an automated analysis instrument such as, for example, a TECHNICON RA-1000 clinical chemistry analyzer. (TECHNICON RA-1000 is a registered trademark of Technicon Instruments Corporation, Tarrytown, NY.)
There are several known substrates for .beta.-galactosidase, including: 2-methoxy-4-(2-nitrovinyl)-phenyl-.beta.-D-galactopyranoside, which was disclosed as a substrate for the assay of .beta.-galactosidase (C. T. Yuen, Analytica Chimica Acta, 163, (1982) 195-204); resorufin-.beta.-D-galactopyranoside, which was disclosed as a fluorogenic substrate for .beta.-galactosidase (Analytica Chimica Acta, 163, (1984) 67-72); E-1-1 (1-deoxylactulosyl)-2-lysine, which was disclosed as a substrate for determining the activity of .beta.-galactosidase in the intestinal tract of mice (J. of Chromatography, 278, (1983) 275-282; and two high molecular weight substrates for .beta.-D-galactosidase, each containing .beta.-D-[3-H]- galactopyranosyl moieties linked through an aliphatic bridge to either poly-2-lysine or polymeric dialdehyde, which were disclosed by R. Madhan et al. (Enzyme, 25 (1980) 127-131).
The most common substrate for 62-galactosidase currently in use is O-nitrophenyl-.beta.-D-galactopyranoside (ONPG) which is preferred for its relatively fast enzyme turnover rate, good stability in aqueous buffers and the relative ease by which it can be synthesized or commercially obtained. ONPG has a significant disadvantage, though, in that it has a relatively small molar absorptivity upon enzyme-catalyzed hydrolysis. Assays for digoxin and other clinically important low concentration analytes which utilize .beta.-galactosidase encapsulated lipsomes require a more sensitive substrate to achieve the essential assay dynamic range.
Therefore, it is desired to produce a chromogenic substrate for .beta.-galactosidase for use in the assay of digoxin and other clinically important low concentration analytes which may be present in serum such as blood, utilizing .beta.-galactosidase-encapsulated liposomes. The substrate should be kinetically equivalent to known substrates such as ONPG and stable in aqueous buffers, but exhibit substantially increased molar absorptivity as compared to known substrates such as ONPG.